The present proposal focuses on exploring the unique structural features of HIV-1 Gag, the principal protein for the viral particle assembly, as a new therapeutic target. In particular, we will test the following hypothesis: interactions of the assembly cofactors (PI(4,5)P2 and RNA) with the Gag polyprotein induce protein conformational changes essential for the assembly of the retroviral particles. The cofactor binding sites and the polyprotein regions responsible for the architectural changes provide attractive targets for developing new antiviral inhibitors. We have set out the following two specific aims. Aim 1. To dissect Gag interactions with the assembly cofactors essential for the viral particle assembly. The following stepwise experimental strategy will be employed to identify PI(4,5)P2 and RNA binding sites and protein-conformational changes in Gag. Initially, our mass spectrometric foot-printing will be employed to reveal certain surface amino acids in Gag that directly coordinate cofactors. These results will be used together with the available structural data on separate Gag domains to create molecular models for the complexes. The contact residues identified from the foot-printing and molecular modeling studies will be mutated. Interactions of the cofactors with the mutant proteins will be analyzed by isothermal titration calorimetry (ITC) and electron microscopy (EM). These studies will enable us to obtain detailed structural information on the binding sites of PI(4,5)P2 and RNA in Gag. In addition, protein-protein interactions triggered by the cofactor binding will be revealed. Taken together, the proposed studies will lead to better understanding of the structural foundations of the viral particle assembly process. Aim 2. To develop and validate a new mass spectrometric methodology for screening of anti-assembly inhibitors. A protein-protein contact revealed in our mass spectrometric analysis will be exploited as a tool to screen the NCI Diversity Set of ~ 2000 compounds. Our assays will employ a biologically relevant Gag:PI(4,5)P2:?RNA complex. Very importantly, these studies can proceed in the absence of further results from Aim1. Rapid analysis and consumption of minute amounts of protein, nucleic acid and inhibitor samples provide a cost effective approach for screening. Potential outcome of these studies include development of a new method that could be exploited for screening of larger chemical libraries to discover novel potent anti-assembly inhibitors. [unreadable] [unreadable] [unreadable]